Fig. 4

Morpholino mediated esco2-knockdown phenotypes include reduction in regenerate length and bone segment length. A: Representative images showing decreased regenerate length in esco2 morpholino (MO) injected half compared with the uninjected (UN) half of the same fin. B: Representative images showing similar levels of regeneration in fins injected with esco2 mismatch (MM) MO compared with the UN half of the same fin. The red line indicates the amputation plane. Arrows indicate the distance from the amputation plane to the distal end of the 3^rd fin ray. C: Representative images of calcein stained segments of the 3^rd fin ray show shorter bone segments in MO-injected fin half compared with the UN half of the same fin. D: Representative images of calcein stained segments of the 3^rd fin ray of MM-injected and UN fins reveal bone segments of similar lengths. For all experiments 6–8 fish were used per trial and at least 3 independent trials were performed. The MO/MM was injected/electroporated in 3 dpa fins and was allowed to regenerate for 4 days. For regenerate length and segment length measurements the fins were calcein stained at 4 dpe/7 dpa. For calcein staining, fish were allowed to swim for 10 min in 0.2% calcein (pH 7) at room temperature followed by fresh system water for another 10 min. E: Graph reveals significant reduction (*) in regenerate length in MO treated fins compared with UN fins. Regenerate length of MM treated fins and UN fins show no significant differences in length. F: Graph shows significant reduction (*) in segment length between MO treated fins and UN fins. The segment length of MM treated fins and UN fins show no significant difference. Student′s t-test was used for determining statistical significance where P < 0.05. Standard error is represented by error bars. Scale bar = 50 µm.